Matrix metalloprotease inhibitors

ABSTRACT

Compounds of the formulas ##STR1## wherein each R 1  is independently H or alkyl (1-8C) and R 2  is alkyl (1-8C) or wherein the proximal R 1  and R 2  taken together are --(CH 2 ) p  -- wherein p=3-5; 
     R 3  is H or alkyl (1-4C); 
     R 4  is fused or conjugated unsubstituted or substituted bicycloaryl methylene; 
     n is 0, 1 or 2; 
     m is 0 or 1; and 
     x is OR 5  or NHR 5 , wherein R 5  is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); or 
     X is an amino acid residue or amide thereof; or 
     X is the residue of a cyclic amine or heterocyclic amine; 
     Y is selected from the group consisting of R 7  ONR 6  CONR 6  -, R 6   2  NCONOR 7  -, and R 6  CONOR 7  -, wherein each R 6  is independently H or lower alkyl (1-4C); R 7  is lower alkyl (1-4C) or an acyl group; and 
     wherein --CONR 3  -- is optionally in modified isoteric form are inhibitors of matrix metalloproteases.

This invention was made with government support under grant HL27368awarded by the National Institutes of Health. The government has certainrights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/615,798, filed Nov. 21, 1990.

TECHNICAL FIELD

The invention is directed to pharmaceuticals which are useful indiseases characterized by unwanted collagenase activity. Morespecifically, the invention concerns substituted or unsubstitutedhydroxyureas and reverse hydroxamates and which include fused orconjugated bicycloaryl substituents.

BACKGROUND ART

There are a number of enzymes which effect the breakdown of structuralproteins and which are structurally related metalloproteases. Theseinclude human skin fibroblast collagenase, human skin fibroblastgelatinase, human sputum collagenase and gelatinase, and humanstromelysin. These are zinc-containing metalloprotease enzymes, as arethe angiotensin-converting enzymes and the enkephalinases.

Collagenase and related enzymes are important in mediating thesymptomology of a number of diseases, including rheumatoid arthritis(Mullins, D. E., et al., Biochim Biophys Acta (1983) 695:117-214); themetastasis of tumor cells (ibid., Broadhurst, M. J., et al., EPapplication 276436 (1987), Reich, R., et al., Cancer Res (1988)48:3307-3312); and various ulcerated conditions. Ulcerative conditionscan result in the cornea as the result of alkali burns or as a result ofinfection by Pseudomonas aeruginosa, Acanthamoeba, Herpes simplex andvaccinia viruses. Other conditions characterized by unwanted matrixmetalloprotease activity include periodontal disease, epidermolysisbullosa and scleritis.

In view of the involvement of collagenase in a number of diseaseconditions, attempts have been made to prepare inhibitors to this enzymeA number of such inhibitors are disclosed in EP applications 126,974(published 1984) and 159,396 (published 1985) assigned to G. D. Searle.These inhibitors are secondary amines which contain oxo substituents atthe 2-position in both substituents bonded to the amino nitrogen.

More closely related to the compounds of the present invention are thosedisclosed in U.S. Pat. Nos. 4,599,361 and 4,743,587, also assigned to G.D. Searle. These compounds are hydroxylamine dipeptide derivatives whichcontain, as a part of the compound, a tyrosine or derivatized tyrosineresidue or certain analogs thereof.

Other compounds that contain sulfhydryl moieties as well as residues ofaromatic amino acids such as phenylalanine and tryptophan are disclosedin PCT application W088/06890. Some of these compounds also containi-butyl side chains.

Inhibitors have also been disclosed for the related protease,thermolysin. These include hydroxamic peptide derivatives described byNishino, N., et al., Biochemistry (1979) 18:4340-4347; Nishino, N., etal., Biochemistry (1978) 17:2846-2850. Tryptophan is also known to betherapeutic in various conditions, some of which may involve collagenase(see, for example, JP 57/058626; U.S. Pat. Nos. 4,698,342; 4,291,048).Also, inhibitors of bacterial collagenases have been disclosed in U.S.Pat. No. 4,558,034.

It has now been found that the compounds described below have superiorinhibiting activity with respect to matrix metalloproteases. Theinvention compounds add to the repertoire of agents available for thetreatment of conditions and diseases which are characterized by unwantedactivity by the class of proteins which destroy structural proteins anddesignated "matrix metalloprotease" herein.

DISCLOSURE OF THE INVENTION

The invention provides new compounds which are useful as inhibitors ofmatrix metalloproteases and which are effective in treating conditionscharacterized by excess activity of these enzymes. In addition, thesecompounds can be used to purify matrix metalloproteases and to detectincreased levels of matrix metalloproteases in vivo. The compounds takeadvantage of the incorporation of tryptophan or other fused orconjugated bicycloaromatic amino acid residues into a substituted orunsubstituted reverse hydroxamate- or a hydroxyurea-derivatized matrixmetalloprotease inhibitor.

Accordingly, in one aspect, the invention is directed to compounds ofthe formula: ##STR2##

wherein each R¹ is independently H or alkyl (1-8C) and R² is alkyl(1-8C) or wherein the proximal R¹ and R² taken together are --(CH₂)_(p)-- wherein p=3-5;

R³ is H or alkyl (1-4C);

R⁴ is fused or conjugated unsubstituted or substituted bicycloarylmethylene;

n is 0, 1, or 2;

m is 0 or 1; and

X is OR⁵ or NHR⁵, wherein R⁵ is H or substituted or unsubstituted alkyl(1-12C), aryl (6-12C), aryl (6-16C); or

X is an amino acid residue or amide thereof; or

X is the residue of a cyclic amine or heterocyclic amine;

Y is selected from the group consisting of R⁷ ONR⁶ CONR⁶ -, R⁶ ₂ NCONOR⁷-, and R⁶ CONOR⁷ -, wherein each R⁶ is independently H or lower alkyl(1-4C); R7 is H, lower alkyl (1-4C) or an acyl group.

Also included within the scope of the invention are compounds whereinthe --CONR3-- amide bond shown is replaced by a modified isosteric bond,such as --CH₂ NR³ --, --CH₂ CHR³ --, --CH═CR³ --, --COCHR³ --,--CHOHCHR³ --, --NR³ CO--, --CF═CR³ --, and the like.

These compounds have the ability to inhibit at least one mammalianmatrix metalloprotease. Accordingly, in other aspects, the invention isdirected to pharmaceutical compositions containing the compounds offormula 1 or 2, to methods of treating diseases characterized by matrixmetalloprotease activity using these compounds or the pharmaceuticalcompositions thereof. Matrix metalloproteases at a particularlyundesired location can be targeted by conjugating the compounds of theinvention to a targeting ligand specific for a marker at that locationsuch as an antibody or fragment thereof or a receptor ligand.

The invention is also directed to various other processes which takeadvantage of the unique properties of these compounds. Thus, in anotheraspect, the invention is directed to the compounds of formulas 1 or 2conjugated to solid supports These conjugates can be used as affinityreagents for the purification of a desired matrix metalloprotease.

In another aspect, the invention is directed to the compounds of formula1 or 2 conjugated to label As the compounds of the invention selectivelybind to at least one matrix metalloprotease, the label can be used todetect the presence of unusually large amounts of matrix metalloproteasein vivo or in vitro cell culture.

In addition, the compounds of formula 1 or 2 can be conjugated tocarriers which permit the use of these compounds in immunizationprotocols to prepare antibodies specifically immunoreactive with thecompounds of the invention. These antibodies are then useful both intherapy and in monitoring the dosage of the inhibitors.

In still another aspect, the invention is directed to methods to preparethe compounds of formulas 1 and 2 and to novel intermediates in theirpreparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the effect of the inhibitor of theinvention on corneal burns using a clinical scoring method.

FIG. 2 shows the corresponding effect of the compound of the inventionon corneal burns using a perforation criterion.

MODES OF CARRYING OUT THE INVENTION

The invention compounds are inhibitors of mammalian matrixmetalloproteases. As used herein, "mammalian matrix metalloprotease"means any enzyme found in mammalian sources which is capable ofcatalyzing the breakdown of collagen, gelatin or proteoglycan undersuitable assay conditions Appropriate assay conditions can be found, forexample, in U.S. Pat. No. 4,743,587, which references the procedure ofCawston, et al., Anal Biochem (1979) 99:340-345, use of a syntheticsubstrate is described by Weingarten, H., et al., Biochem Biophys ResComm (1984) 139:1184-1187. Any standard method for analyzing thebreakdown of these structural proteins can, of course, be used. Thematrix metalloprotease enzymes referred to in the herein invention areall zinc-containing proteases which are similar in structure to, forexample, human stromelysin or skin fibroblast collagenase.

The ability of candidate compounds to inhibit matrix metalloproteaseactivity can, of course, be tested in the assays described above.Isolated matrix metalloprotease enzymes can be used to confirm theinhibiting activity of the invention compounds, or crude extracts whichcontain the range of enzymes capable of tissue breakdown can be used.

The invention compounds can be considered to comprise two componentslinked by an amide or modified amide bond. One component is substitutedor unsubstituted hydroxyurea or reverse hydroxamate derivative of adicarboxylic acid backbone, wherein the alternate carboxyl group formsthe amide or modified bond with an amino acid or analog thereof. Theamino acid or analog is the residue of an amino acid or analog whichcontains a fused or conjugated bicycloaromatic system, such as atryptophan residue or a naphthylalanyl residue. This residue can also beamidated or can be extended by one or two additional amino acidresidues. Thus, the compounds of the invention can be prepared by themethods described below.

The dicarboxylic acid residue in formula 1 contains at least one and, insome instances, two or more chiral centers. Either configuration at anychiral center is included within the invention, as are mixtures ofcompounds containing the two possible configurations at each point ofchirality. However, it is generally found that a particularconfiguration at each of these chiral centers is preferred. Similarly,in the compounds of formula 2, the double bond can be either the cis ortrans configuration. In this case, also, one or the other configurationfor a particular set of embodiments will be preferred. The carbon towhich R⁴ is bound is chiral in both formulas. While both configurationsare included in the invention, that corresponding to an L-amino acid ispreferred.

The amide bond shown as --CONR³ -- in the compounds of formulas 1 and 2may be in "modified isosteric" form. Compounds of the invention wherethis is the case are preferred when oral administration is desirable. By"modified isosteric form" is meant that in lieu of the functionality--CONR³ --, the compound has instead a moiety such as those selectedfrom the group consisting of --CH₂ NR³ --, --CH--NR³, --COCHR³ --,--CH(OH)NR³, --CH(OH)CHR³ --, --CSCHR³ --, --CH═CR³ --, CF═CR³ and --NR³CO--.

As used herein, "alkyl" has its conventional meaning as a straightchain, branched chain or cyclic saturated hydrocarbyl residue such asmethyl, ethyl, isobutyl, cyclohexyl, t-butyl or the like. The alkylsubstituents of the invention are of the number of carbons noted whichmay be substituted with 1 or 2 substituents. Substituents are generallythose which do not interfere with the activity of the compound,including hydroxyl, "CBZ," amino, and the like. Aryl refers to aromaticring systems such as phenyl, naphthyl, pyridyl, quinolyl, indolyl, andthe like; aryl alkyl refers to aryl residues linked to the positionindicated through an alkyl residue In all cases the aryl portion may besubstituted or unsubstituted. "Acyl" refers to a substituent of theformula RCO- wherein R is alkyl or arylalkyl as above-defined. Thenumber of carbons in the acyl group is generally 1-15; however, as theacyl substituent is readily hydrolyzed in vivo, the nature of the groupis relatively unimportant. "Cyclic amines" refer to those amines wherethe nitrogen is part of a heterocyclic ring, such as piperidine,"heterocyclic amines" refer to such heterocycles which contain anadditional heteroatom, such as morpholine.

In the compounds of formula 1, preferred embodiments for R¹ and R²include those wherein each R¹ is H or Me and R² is alkyl of 3-8C,especially isobutyl, 2-methyl butyl, or isopropyl. Especially preferredis isobutyl. Preferred also are those compounds of formula 1 or 2wherein n=1 or m=1.

In both formula 1 and 2 compounds, preferred embodiments of R³ are H andmethyl, especially H.

R⁴ is a fused or conjugated bicyclo aromatic system linked through amethylene group to the molecule. By "fused or conjugated bicycloaromatic system" is meant a two-ringed system with aromatic characterwhich may, further, contain one or more heteroatoms such as S, N, or O.When a heteroatom such as N is included, the system as it forms a partof formula (1) or (2), may contain an acyl protecting group (1-5C)attached to the nitrogen. Representative bicyclo fused aromatic systemsinclude naphthyl, indolyl, quinolinyl, and isoquinolinyl. Representativeconjugated systems include biphenyl, 4-phenylpyrimidyl, 3-phenylpyridyland the like. In all cases, any available position of the fused orconjugated bicyclic system can be used for attachment through themethylene. The fused or conjugated aromatic system may further besubstituted by 1-2 alkyl (1-4C) residues and/or hydroxy or any ringnitrogens may be acylated. Preferred acylation is acetylation.

Preferred embodiments of R⁴ include 1-(2-methyl naphthyl)methylene;1-quinolyl methylene; 1-naphthyl methylene; 2-naphthyl methylene;1-isoquinolyl methylene; 3-isoquinolyl methylene; 3-thionaphthenylmethylene; 3-cumaronyl methylene; 3-(5-methylindolyl)methylene;3-(5-hydroxyindolyl)methylene; 3-(2-hydroxyindolyl)-methylene; biphenylmethylene; and 4-phenylpyrimidyl methylene; and the substituted formsthereof.

Many of these substituents as part of an amino acid residue aredescribed in Greenstein and Winitz, "Chemistry of the Amino Acids"(1961) 3:2731-2741 (John Wiley & Sons, N.Y.).

A particularly preferred embodiment of R⁴ is 3-indolyl methylene or itsN-acylated derivative--i.e., that embodiment wherein the "C-terminal"amino acid is a tryptophan residue or a protected form thereof. Apreferred configuration at the carbon to which R⁴ is bound is thatcorresponding to L-tryptophan.

Preferred embodiments of X are those of the formula NHR⁵ wherein R⁵ isH, substituted or unsubstituted alkyl (1-12C) or aryl alkyl (6-12C).Particularly preferred substitutions on R⁵ are a hydroxyl group, or aphenylmethoxycarbamyl (CBZ) residue. In addition, the compound may beextended by embodiments wherein X is an additional amino acid residue,particularly a glycyl residue, which may also be amidated as described.

Therapeutic Use of the Compounds of the Invention

As set forth in the Background section above, a number of diseases areknown to be mediated by excess or undesired matrix-destroyingmetalloprotease activity. These include tumor metastasis, rheumatoidarthritis, skin inflammation, ulcerations, particularly of the cornea,reaction to infection, and the like. Thus, the compounds of theinvention are useful in therapy with regard to conditions involving thisunwanted activity.

The invention compounds can therefore be formulated into pharmaceuticalcompositions for use in treatment or prophylaxis of these conditions.Standard pharmaceutical formulation techniques are used, such as thosedisclosed in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., latest edition.

For indications to be treated systemically, it is preferred that thecompounds be injected. These conditions include rheumatoid arthritis andtumor metastasis. The compounds can be formulated for injection usingexcipients conventional for such purpose such as physiological saline,Hank's solution, Ringer's solution, and the like. Injection can beintravenous, intramuscular, intraperitoneal or subcutaneous. Dosagelevels are of the order of 0.1 μg/kg of subject to 1 mg/kg of subject,depending, of course, on the nature of the condition, the nature of thesubject, the particular embodiment of the invention compounds chosen,and the nature of the formulation and route of administration.

In addition to administration by injection, the compounds of theinvention can also be formulated into compositions for transdermal ortransmucosal delivery by including agents which effect penetration ofthese tissues, such as bile salts, fusidic acid derivatives, cholicacid, and the like. The invention compounds can also be used inliposome-based delivery systems and in formulations for topical and oraladministration depending on the nature of the condition to be treated.Oral administration is especially advantageous for those compoundswherein the moiety --CONR³ -- is in a modified isosteric form. Thesecompounds resist the hydrolytic action of the digestive tract. Oralformulations include syrups, tablets, capsules, and the like, or thecompound may be administered in food or juice.

The inhibitors of the invention can be targeted to specific locationswhere the matrix metalloprotease is accumulated by using targetingligands. For example, to focus the inhibitors to matrix metalloproteasecontained in a tumor, the inhibitor is conjugated to an antibody orfragment thereof which is immunoreactive with a tumor marker as isgenerally understood in the preparation of immunotoxins in general. Thetargeting ligand can also be a ligand suitable for a receptor which ispresent on the tumor. Any targeting ligand which specifically reactswith a marker for the intended target tissue can be used. Methods forcoupling the invention compound to the targeting ligand are well knownand are similar to those described below for coupling to carrier. Theconjugates are formulated and administered as described above.

For localized conditions, topical administration is preferred. Forexample, to treat ulcerated cornea, direct application to the affectedeye may employ a formulation as eyedrops or aerosol. For cornealtreatment, the compounds of the invention can also be formulated as gelsor ointments, or can be incorporated into collagen or a hydrophilicpolymer shield. The materials can also be inserted as a contact lens orreservoir or as a subconjunctival formulation. For treatment of skininflammation, the compound is applied locally and topically, in a gel,paste, salve or ointment. The mode of treatment thus reflects the natureof the condition and suitable formulations for any selected route areavailable in the art.

Particular topical conditions that are susceptible to treatment usingthe compounds of the invention include stomach ulcers, superficialwounds of any type, epidermolysis bullosa, various forms of skin cancer,and pemphigus. It is well known that collagenase is involved in theprogress of stomach ulcers, as described by Hasabe, T., et al., J ExpClin Med (1987) 12:181-190; Ozaki, I., et al., Scand J GastroenterolSuppl (Norway) (1989) 16:138-141. Collagenase is also found in marginalwound tissue as reviewed by Mullins, D. E., et al., Biochim Biophys Acta(1983) 695:177-214, and patients showing impaired wound healing areknown to have increased collagenase activity in the wound tissue. Forexample, Hennesey, P. J., et al., J Pediat Surg (1990) 25 25:75-78,showed this to be the case in diabetes; Sank, A , et al., Surgery (1989)106:1141-1148, demonstrated this enhanced collagenase activity inparaplegia, chronic renal failure, and Cushings disease. Thus, thecollagenase inhibitors of the invention are useful in any ulcerativeskin condition, including, for example, decubitus ulcers, or otherconditions where wound healing is slow; other conditions susceptible totreatment by the compounds of the invention include corneal or scleralmelting associated with keratomalacia, scleromalacia perforans andconnective tissue diseases.

This is also the case with respect to wounds inflicted in the course ofmedical treatment. It has been demonstrated that collagenase inhibitorsenhance the strength of suture lines and suture holding capacity inintestinal anastomoses in rats (Hogstrom, H., et al., Res Exp Med (1985)185:451-455. Other topical conditions responsive to the collagenaseinhibitors of the invention include epidermolysis bullosa whereincreased collagenase activity has been demonstrated (Perez-Tamayo, R.,Am J Path (1978) 92:509-566, pp. 539-541). It is also known thatcollagenase activity is increased in the stroma surrounding basal cellcarcinoma and throughout tumor stromas in ulcerated basal cell carcinoma(Childer, S., J. W., et al., J Am Acad Dermatol (1987) 17:1025-1032).

Further, the matrix metalloprotease inhibitors of the invention areuseful in the treatment of septic shock and in the treatment of adultrespiratory distress syndrome (ARDS). The role of plasma proteases inseptic shock has been shown by Colman, R. C., New Eng J Med (1989)320:1207-1210, and the participation of neutrophil proteases in bothseptic shock and ARDS has been shown by Idell, S., et al., Ann ResRespir Dis (1985) 132:1098-1105. In the case of these conditions, thecompounds of the invention protect the connective tissue directly fromdigestion by matrix metalloproteases as well as prevent the degradationof collagen and therefore prevention of known chemotaxis of neutrophilstoward collagen fragments (Werb, Z., in "Textbook of Rheumatology,"Kelley, W. N., et al., eds. (1989) W. B. Saunders, Philadelphia, 3rded., pp. 300-320). The compounds of the invention also prevent theinactivation of plasma protease inhibitors by matrix metalloproteases(Werb, Z. supra).

In all of the foregoing, of course, the compounds of the invention canbe administered alone or as mixtures, and the compositions may furtherinclude additional drugs or excipients as appropriate for theindication.

Some of the compounds of the invention also inhibit bacterialmetalloproteases although generally at a lower level than that exhibitedwith respect to mammalian metalloproteases. Some bacterialmetalloproteases seem to be less dependent on the stereochemistry of theinhibitor, whereas substantial differences are found betweendiastereomers in their ability to inactivate the mammalian proteases.Thus, this pattern of activity can be used to distinguish between themammalian and bacterial enzymes.

Preparation and Use of Antibodies

The invention compounds can also be utilized in immunization protocolsto obtain antisera immunospecific for the invention compounds. As theinvention compounds are relatively small haptens, they areadvantageously coupled to antigenically neutral carriers such as theconventionally used keyhole limpet hemocyanin (KLH) or serum albumincarriers. Coupling to carrier can be done by methods generally known inthe art; the --COX functionality of the invention compounds offers aparticularly convenient site for application of these techniques. Forexample, the COX residue can be reduced to an aldehyde and coupled tocarrier through reaction with sidechain amino groups in protein-basedcarriers, optionally followed by reduction of imino linkage formed. TheCOX residue wherein X=OH can also be reacted with sidechain amino groupsusing condensing agents such as dicyclohexyl carbodiimide or othercarbodiimide dehydrating agents. Linker compounds can also be used toeffect the coupling; both homobifunctional and heterobifunctionallinkers are available from Pierce Chemical Company, Rockford, Ill.Compounds 31-34 described in the examples below are designed to becoupled to antigenically neutral carriers through their C-terminalcarboxyl groups (or amino groups in compound 32) using appropriatecoupling agents.

The resulting immunogenic complex can then be injected into suitablemammalian subjects such as mice, rabbits, and the like. Suitableprotocols involve repeated injection of the immunogen in the presence ofadjuvants according to a schedule which boosts production of antibodiesin the serum. The titers of the immune serum can readily be measuredusing immunoassay procedures, now standard in the art, employing theinvention compounds as antigens.

The antisera obtained can be used directly or monoclonal antibodies maybe obtained by harvesting the peripheral blood lymphocytes or the spleenof the immunized animal and immortalizing the antibody-producing cells,followed by identifying the suitable antibody producers using standardimmunoassay techniques.

The polyclonal or monoclonal preparations are then useful in monitoringtherapy or prophylaxis regimens involving the compounds of theinvention. Suitable samples such as those derived from blood, serum,urine, or saliva can be tested for the presence of the administeredinhibitor at various times during the treatment protocol using standardimmunoassay techniques which employ the antibody preparations of theinvention.

The invention compounds can also be coupled to labels such asscintigraphic labels, e.g., technetium 99 or I-131, using standardcoupling methods. The labeled compounds are administered to subjects todetermine the locations of excess amounts of one or more matrixmetalloproteases in vivo. The ability of the inhibitors to selectivelybind matrix metalloprotease is thus taken advantage of to map thedistribution of these enzymes in situ. The techniques can also, ofcourse, be employed in histological procedures and the labeled inventioncompounds can be used in competitive immunoassays.

Use As Affinity Ligands

The invention compounds can be coupled to solid supports, such asseparation membranes, chromatographic supports such as agarose,sepharose, polyacrylamide, and the like, or to microtiter plates toobtain affinity supports useful in purification of various mammalianmatrix metalloproteases. The selective binding of the matrixmetalloproteases to the inhibitor ligand permits the adsorption of thedesired enzyme and its subsequent elution using, for example, alteredionic strength and/or pH conditions.

Preparation of the Invention Compounds

The reverse hydroxamates and hydroxyureas are more stable biologicallythan the corresponding hydroxamates per se. This has been confirmed inCarter, G. W., et al., J Pharmacol Exp Ther (1991) 256:929-937; Jackson,W. P., et al., J Med Chem (1988) 31:499-500; Young, P. R., et al., FASEBJ (1991) 5:A1273; Hahn, R. A., et al., J Pharmacol Ex Ther (1991)256:94-102; Tramposch, K. M., et al., Agents Actions (1990) 30:443-450;Argentieri, D. C., et al.; Kimball, E., et al., 5th Int ConfInflammation Research Assoc., Whitehaven, Pa., Sep. 23-27, 1990,Abstract 100; and Huang, F., et al., J Med Chem (1989) 32:1836-1842.Thus, while somewhat more complicated to synthesize, these analogs offerphysiological characteristics which are advantageous in the applicationsof these compounds to therapy.

The reverse hydroxamates and hydroxyureas of the invention areobtainable using the standard techniques of synthetic organic chemistry(see Challis, B. C., et al., "Amides and Related Compounds" in"Comprehensive Organic Chemistry," Barton, D., et al., eds. (1979)2:1036-1045), Pergamon Press, Oxford, as further described below.

With respect to starting materials, the components forming the --NR³--CHR⁴ COX moiety are readily available in the case of tryptophan andits analogs as esters or amides. As set forth above, many analogousfused bicyclo aromatic amino acids are described by Greenstein andWinitz (supra) Amino acids corresponding to those wherein R⁴ is1-(2-methyl naphthyl)methylene; 1-quinolyl-methylene; 1-naphthylmethylene; 1-isoquinolyl methylene; and 3-isoquinolyl methylene can beprepared from the bicyclo aromatic methylene halides using the acetamidomalonic ester synthesis of amino acids, as is well understood in theart. The methylene halides themselves can be prepared from theircorresponding carboxylic acids by reduction with lithium aluminumhydride and bromination of the resulting alcohol with thionyl bromide.

Depending on the functional group symbolized by Y, the stage ofsynthesis at which this moiety is brought into the compound of theinvention varies.

For those embodiments wherein Y is R⁷ ONR⁶ CONR⁶ -- and wherein n=0, 1or 2, the compounds are prepared by acylating an α, β or γ amino acid,respectively with methyl or ethyl chloroformate, condensing theresulting amino acid with a protected form of the moiety --NR³ CHR⁴ COXand reacting the resulting carboethoxy "dipeptide" with hydroxylamine ora substituted hydroxylamine as described by Fieser, L. F., et al.,"Reagents for Organic Synthesis" (1967) 1:479 (John Wiley & Sons, NewYork). This sequence of reactions is shown in Reaction Scheme 1.##STR3##

Alternatively, the α, β or γ amino acid is temporarily protected using,for example, carbobenzoxy or tertiary butyloxycarbonyl and coupling itto the carboxy-terminal-protected amino acid moiety containing R⁴. Theprotecting group is then removed by hydrogenolysis or acidolysis asappropriate, and the deprotected α, β or γ amino group is reacted withan activated carbonic acid such as carbonyldiimidazole. The resultant isthen reacted with hydroxylamine or substituted hydroxylamine to obtainthe desired product. This sequence of reactions is summarized inReaction Scheme 2. (In the formula Im-Co-Im, Im represents an immidazoleresidue.) ##STR4##

The appropriate α, β or γ amino acids are prepared by general methods asset forth by Jones, J. H., et al., in "Amino Acids," p. 834 (Barton, D.,et al., eds.) ("Comprehensive Organic Chemistry" (1979) Vol. 2, PergamonPress) Such methods include, for example, homologation by Arndt-Eistertsynthesis of the corresponding N-protected o-amino acid and moregenerally the addition of nitrogen nucleophiles such as phthalimide toα,β-unsaturated esters, acids or nitriles.

In a second class of hydroxyureas, Y has the formula R⁶ ₂ NCONOR⁷ - andn is 0, 1 or 2. These compounds are prepared from the corresponding α, βor γ hydroxyamino acids of the formula R⁷ ONH(CHR¹)_(n) CHR² COOH. Whenboth R⁶ are H, this intermediate is converted to the desired hydroxyureaby reaction with silicon tetraisocyanate, as described by Fieser andFieser, "Reagents for Organic Synthesis" (1968) 1:479 (John Wiley &Sons, New York). The reaction is conducted with the hydroxyl groupprotected or substituted by R⁷. The resulting hydroxyurea is thencoupled to the component of the formula HNR³ CHR⁴ COX to obtain thedesired product. Alternatively, the amide is first formed and theN-hydroxyl dipeptide is treated with the reagent.

Alternatively, when Y is R⁶ HNCO-NOR⁷, wherein R⁶ is alkyl, the aboveO-protected α, β or γ N-hydroxyamino acid is reacted with the relevantalkylisocyanate R⁶ NCO to produce the desired product.

When Y is of the formula R⁶ ₂ NCO-NOR⁷ - wherein both R⁶ are alkyl, theα, β or γ N-hydroxyamino acid is reacted with an activated form ofcarbonic acid, for example, carbonyldiimidazole orbis-p-nitrophenylcarbonate, and then with the diamine R⁶ ₂ NH whereinboth R⁶ are alkyl groups. This is followed by deprotection, if desired.

Conditions for the foregoing can be found in the descriptions ofanalogous preparations for tripeptides as described by Nishino, N., etal., Biochemistry (1979) 18:4340-4346.

The β-N-hydroxyamino acids used as intermediates in the foregoingsynthesis can be prepared by a malonic ester synthesis in which diethylmalonate is alkylated twice, one with R² -Br and then withbenzylchloromethyl ether, for example, for the case wherein R¹ is H. Theproduct is saponified, decarboxylated, hydrogenated, and oxidized togive the β-aldehyde in a manner similar to the synthesis of a homologousaldehyde described by Kortylewicz, Z. P., et al., Biochemistry (1984)23:2083-2087. The desired β-hydroxyamino acid is then obtained byaddition of protected (or alkylated, if R⁷ is alkyl or acylated if R7 isacyl) hydroxylamine. The corresponding compound wherein R¹ is alkyl canbe prepared in an analogous manner wherein the second alkylationutilizes benzyl-O-CHR¹ Cl. The homologous ketone was described byGalardy, R. E., al., Biochemistry (1985) 24:7607-7612.

Finally, those compounds wherein Y is of the formula R⁶ CONOR⁷ -, i.e.,the reverse hydroxymates, can be prepared by acylation of thecorresponding α, β or γ N-hydroxy dipeptide. Alternatively, theN-hydroxyamino the amide bond in the compounds of the invention. Theacylation method is described by, for example, Nishino, N., et al.,Biochemistry (1979) 18:4340-4346, cited above.

Alternatively, for those compounds wherein n=1 and R¹ is H, thecompounds can be prepared by condensing the ylide1,1-dimethoxy-2-(triphenylphosphoranylidene) ethane prepared fromtriphenylphosphine and 1,1-dimethoxy-2-bromoethane with4-methyl-2-oxopentanoic acid. The product is then hydrogenated to obtain4,4-dimethoxy-2-isobutylbutanoic acid which is coupled to the moiety R³NHCHR⁴ COX to obtain 4,4-dimethoxy-2-isobutylbutanoyl-NR³ CHR⁴ COX.Treatment with aqueous acid yields the aldehyde2-isobutyl-4-oxobutanoyl-NR³ CHR⁴ COX. The oxime is prepared by reactionwith hydroxylamine and reduced to the corresponding N-substitutedhydroxylamine. Acylation of both the hydroxaminol oxygen and nitrogenfollowed by hydrolysis of the O-acyl group provides the N-acyl reversehydroxymates. (Summers, J. B., et al., J Med Chem (1988) 31:1960-1964.)

For compounds wherein --CONR³ -- is in modified isosteric form, theseforms can be prepared by methods known in the art. The followingreferences describe preparation of peptide analogs which include thesealternative-linking moieties: Spatola, A. F. Vega Data (March, 1983),Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review);Spatola, A. F., in "Chemistry and Biochemistry of Amino Acids Peptidesand Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983) (genera) review); Morley, J. S., Trends Pharm Sci (1980) pp.463-468 (general review); Hudson, D., et al., Int J Pept Prot Res (1979)14:177-185 (--CH₂ NR³ --, --CH₂ CHR³ --); Spatola, A. F., et al., LifeSci (1986) 38:1243-1249 (--CH₂ --S); Hann, M. M., J Chem Soc PerkinTrans I (1982) 307-314 (--CH--CR³ --, cis and trans); Almquist, R. G.,et al., J Med Chem (1980) 23:1392-1398 (--COCHR³ --); Jennings-White,C., et al., Tetrahedron Lett (1982) 23:2533 (--COCHR³ --); Szelke, M ,et al., European Application EP 45665 (1982) CA:97:39405 (1982)(--CH(OH)CHR³ --); Holladay, M. W., et al., Tetrahedron Lett (1983)24:4401-4404 (--C(OH)CH₂ --); and Hruby, V. J., Life Sci (1982)31:189-199 (--CH₂ --S--).

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLES

In the examples below, TLC solvent systems are as follows: (A) ethylacetate/methanol (95:5); (B) ethyl acetate/methanol (25:5); (C) ethylacetate; (D) ethyl acetate/methanol (30:5); (E) ethyl acetate/hexane(1:1); (F) chloroform/methanol/acetic acid (30:6:2); (G)chloroform/methanol/acetic acid (85:10:1).

Example 1 Preparation ofN-[D,L-2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-tryptophanmethylamide

A suspension of 5 g (0.033 mol) of the sodium salt of4-methyl-2-oxopentanoic acid and 5.65 g (0.033 mol) of benzyl bromide in10 ml of anhydrous dimethylformamide was stirred for 4 days at roomtemperature. After evaporation of the solvent under reduced pressure theresidue was diluted to 100 ml with hexane and washed with water (3×20ml) and saturated sodium chloride and dried over anhydrous magnesiumsulfate. Evaporation of solvent gave 6.4 g (88% yield) of the benzylester of 4-methyl-2-oxopentanoic acid (1) as a colorless oil.

A mixture of 6.4 g (0.029 mol) of (1) and 9.7 g (0.029 mol) ofmethyl(triphenylphosphoranylidene)acetate in 100 mL of dry methylenechloride was stirred for 12 hr at room temperature and evaporated todryness. The residue was extracted with hexane (3×50 mL). The hexanesolution was washed with 10% sodium bicarbonate (2×30 mL), water andsaturated sodium chloride and dried over anhydrous magnesium sulfate.Evaporation of the solvent gave 8.01 g (100% yield) of benzyl2-isobutyl-3-(methoxycarbonyl)-propionate (2) as a mixture of E and Zisomers.

A mixture of 8.01 g (0.029 mol) of (2) and 1 g of 10% palladium oncarbon in 50 mL of methanol was hydrogenated at room temperature under 4atmospheres of hydrogen gas for 8 hr. After removal of the catalyst byfiltration the filtrate was evaporated to dryness under reduced pressureto give 4.7 g (86% yield) of 2-isobutyl-3-(methoxycarbonyl)-propionicacid (3) as a colorless oil.

To a mixture of 0.85 g (4.5 mmol) of (3) and 0.57 g (4.5 mmol) of oxalylchloride in 10 mL of dry methylene chloride 0.1 mL of anhydrousdimethylformamide was added. After stirring for 1 hr at room temperaturethe solvent was evaporated under reduced pressure and the residue wasdiluted to 5 mL with anhydrous dimethylformamide and 1.06 g (4.1 mmol)of the hydrochloride salt of L-tryptophan methylamide (Kortylewicz andGalardy, J Med Chem (1990) 33:263-273) was added followed by addition of1.3 mL (9.3 mmol) of triethylamine at -10° C. This was stirred for 7 hrat room temperature and evaporated to dryness at room temperature underreduced pressure. The residue was diluted to 150 mL with ethyl acetateand washed with water (2×15 mL), 10% potassium bisulfate (5×20 mL), 10%sodium bicarbonate (2×20 mL), saturated sodium chloride and dried overanhydrous magnesium sulfate and then evaporated to give 1.6 g (83%yield) of N-[D,L-2-isobutyl-3-(methoxycarbonyl)propanoyl]-L-tryptophanmethylamide 4 as a mixture of diastereomers, 4A and 4B.

Isomers 4A and 4B were separated by flash chromatography (silica gel,ethyl acetate).

Isomer 4A: mp=134°-137° C. R_(f) (C)=0.37.

Isomer 4B: mp=156°-158° C. R_(f) (C)=0.2.

Alternatively, the mixture of 4A and 4B was converted directly to itshydroxamate as described below. In this case, 5A was crystallized fromthe mixture of 5A and 5B.

A warm mixture of 0.22 g (3.96 mmol) of potassium hydroxide in 1 mL ofmethanol was added to a warm mixture of 0.184 g (2.65 mmol) of thehydrochloride salt of hydroxylamine. After cooling in ice under an argonatmosphere the potassium chloride was filtered off and 0.5 g (1.32 mmol)of (4A) was added to the filtrate. The resulting mixture was stirred for7 hr at room temperature and evaporated to dryness under reducedpressure. The residue was suspended in 100 mL of ethyl acetate andwashed with 10 mL of 10% potassium bisulfate, saturated sodium chlorideand dried over anhydrous magnesium sulfate and evaporated to drynessunder reduced pressure. The residue was crystallized from ethyl acetateto give 0.28 g (56% yield) of pure 5A.

Isomer 4B was converted to its corresponding hydroxamic acid 5B (72%yield) as described for 4A.

Isomer 5A: mp=176°-182° C. R_(f) (D)=0.45.

Isomer 5B: mp=157°-162° C. R_(f) (D)=0.39.

For the case wherein the 4A/4B mixture is used, the 5A can becrystallized directly from the residue as described above.

In a similar manner to that set forth above, but substituting for4-methyl-2-oxopentanoic acid, 2-oxopentanoic acid, 3-methyl-2-oxobutyricacid, 2-oxohexanoic acid, 5-methyl-2-oxohexanoic acid, or 2-decanoicacid, the corresponding compounds of formula 1 are prepared wherein R¹is H and R² is an n-propyl, i-propyl, n-butyl, 2-methylbutyl, andn-octyl, respectively. In addition, following the procedures set forthhereinabove in Example 1, but omitting the step of hydrogenating theintermediate obtained by the Wittig reaction, the correspondingcompounds of formula 2 wherein R¹ is H and R² is as set forth above areobtained

To synthesize the compounds containing acylated forms of the indolylresidue, the intermediate ester of formula 3 or 4 is deesterified andacylated prior to conversion to the hydroxamate. For illustration, 4A isdeesterified with sodium hydroxide in ethanol and then acidified to giveN-(L-2-isobutyl-3-carboxypropanoyl)-L-tryptophan methylamide, which istreated with the anhydride of an alkyl (1-4C) carboxylic acid to obtainN-(L-2-isobutyl-3-carboxypropanoyl)-L-((N-acyl)indolyl)-tryptophanmethylamide. This intermediate is then treated with oxalyl chloridefollowed by hydroxylamine at low temperature to give the correspondinghydroxamate.

Example 2 Preparation ofN-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-D-tryptophanmethylamide (7B)

The mixture of the two diastereoisomers ofN-[2-isobutyl-3-(methoxycarbonyl)-propanoyl]-D-tryptophan methyl amide6A,B was prepared as described for 4A,B in Example 1. The mixture wascrystallized from ethyl acetate to give, after two recrystallizations,0.26 g (49%) of the pure diastereomer 6B: mp 155°-157° C., R_(f)(C)=0.32. 6B was converted into its hydroxamic acid 7B by the methoddescribed in Example 1 in 50% yield (119 mg): mp 157°-159° C., R_(f)(D)=0.39.

Example 3 Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-N-methyl-L-tryptophan methylamide (9A)

The reaction of N-methyl-L-tryptophanmethylamide, prepared as describedin Example 1 for L-tryptophan methylamide, with 3 performed as describedfor 4 gave crudeN-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-N-methyl-L-tryptophanmethylamide 8A,B which was crystallized from ethyl acetate to give 76 mg(19% yield) of 8A: mp 171°-174° C., R_(f) (C)=0.40.

8A was converted into 9A by the method described in Example 1 in 45%yield (34 mg): mp 180°-183° C., R_(f) (D)=0.54.

Example 4 Preparation of N-[2-isobutyl-3-(N-hydroxycarbonylamido)-propanoyl]-L-3-(2-naphthyl)-alanine methylamide (11A)

N-[D,L-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-3-(2-naphthyl)-alanine10A was prepared as described in Example 1 from L-3-(2-naphthyl)-alaninemethylamide and 3. The crude product was chromatographed on 60 g ofsilica gel in ethyl acetate:hexane 1:1 to yield 12 mg (5% yield) of 10A:mp 151°-158° C., R_(f) (C)=0.69.

10A was converted into the hydroxamate 11A as in Example 1 in 30% yield(3 mg): mp 179°-181° C., R_(f) (D)=0.17. MS-FAB (m/z) 400 (M⁺ +H).

Example 5 Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan 2-hydroxyethylamide (13A)

The hydrochloride salt of L-tryptophan 2-hydroxyethylamide was preparedand coupled with 3 as described for the hydrochloride salt ofL-tryptophan methylamide in Example 1 except that 3 was activated with1,1'-carbonyldiimidazole for 20 minutes in methylene chloride at roomtemperature. The crude product was a mixture of 0.7 g (67% yield) of thediastereoisomers 12A,B: R_(f) (C) 12A 0.38, R_(f) (C) 12B 0.19.

12A crystallized from ethyl acetate in 35% yield (0.18 g): mp 161°-163°C., R_(f) (C)=0.38.

12A was converted intoN-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan2-hydroxyethylamide 13A as in Example 1 in 35% yield (62 mg): R_(f)(D)=0.17, mp 162°-163° C. MS-FAB (m/z) 419 (M⁺ +H).

Example 6 Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan amylamide (15A)

The hydrochloride salt of L-tryptophan amylamide was prepared asdescribed in Example 1 for L-tryptophan methylamide and was reacted with3 that had been activated with 1,1'-carbonyldiimidazole for 20 minutesin dichloromethane at room temperature. The mixture of the twodiastereomers ofN-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-tryptophan amylamide14A,B (90% yield) was converted to its corresponding hydroxamic acids asdescribed for 4A. Slow evaporation of the ethylacetate solution gave0.343 g (71%) of 15A,B: mp 160°-163° C. MS-FAB (m/z) 445 (M⁺ +H).

Example 7 Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan piperidinamide (17A,B)

L-tryptophan piperidinamide was reacted with 3 as performed in Example 1for L-tryptophan methylamide to give 1.14 g (89% yield) ofN-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-tryptophanpiperidinamide 16A,B as a foam; R_(f) (C) (16A) 0.74, (16B) 0.67.

16A,B was converted into crude 17A,B identically to 4A in Example 1 in88% yield (570 mg): R_(f) (D) (17A) 0.41, (17B) 0.30. Crude 17A,B waschromatographed on 180 g of silica gel in 12% isopropanol in ethylacetate to give 140 mg (25% yield) of 17A,B after crystallization fromethyl acetate: mp 169°-170° C. MS-FAB (m/z) 443 (M⁺ +H).

Example 8 Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan dodecylamide (19A)

The reaction of L-tryptophan dodecylamide was prepared in a manneranalogous to that described for L-tryptophan methylamide in Example 1.This ester was reacted with 3 as described in Example 1 to give crudeN-[D,L-isobutyl-3-(methoxycarbonyl)-propanol]-L-tryptophan dodecylamide18A,B in 93% yield as a mixture of isomers 19A and 19B. This mixture waschromatographed on 150 g of silica gel in ethyl acetate:hexane, 1:2, toyield 0.62 g of the mixture of the two isomers: R_(f) (E) 19A 0.37,R_(f) (E) 19B 0.29.

Crystallization by slow evaporation from ethyl acetate gave 0.38 g of18A contaminated by approximately 10% of 18B by TLC and NMR analysis: mp133-°135° C. 18A was converted to its corresponding hydroxamic acid asdescribed in Example 1, except that the potassium salt of 19Acrystallized from the alkaline reaction mixture in 81% yield (222 mg).The potassium salt of 19A (54 mg) was dissolved in 2 mL of boilingmethanol, a few drops of water were added, and the solution wasacidified to pH 6 with 0.1N hydrochloric acid and diluted with water togive 50 mg (100% yield) of 19A: mp 155°-159° C., R_(f) (D)=0.49. MS-FAB(m/z) 543 (M⁺ +H).

Example 9 Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)propanoyl]-L-tryptophan (S)-methylbenzylamide (21A)

The reaction of L-tryptophan (S)-methylbenzylamide with 3 was performedas described in Example 1 to give, after crystallization from ethylacetate, 330 mg (51% yield) ofN-[2-isobutyl-3-(methoxycarbonyl)propanoyl]-L-tryptophan(S)-methylbenzylamide 20A: mp 160°-162° C., R_(f) (C)=0.77.

20A was converted into hydroxamate 21A by the identical method used inExample 1 in 38% yield (76 mg): mp 165°-166° C., R_(f) (D)=0.73. MS-FAB(m/z) 479 (M⁺ +H).

Example 10 Preparation ofN-[L-2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan(6-phenylmethoxycarbonylamino-hexyl-1)amide (27A)

To prepare 1-amino-6-phenylmethoxycarbonylamino-hexane (23), anequimolar mixture (0.01 mol) of 1,6-diaminohexane and benzaldehyde in 25mL of methylene chloride was stirred for 5 hr in the presence of 1.5 gof anhydrous magnesium sulfate at room temperature. After removing thedrying agent by filtration the filtrate was evaporated to dryness underreduced pressure to give 2 g (100% yield) of crude1-amino-6-phenylamino-hexane 22 as a colorless oil; NMR(CDCl₃)1.1-1.9(m, 10H, hexane CH₂ -2,-3,-4,-5, NH₂); 2.6(m, 2H, CH₂ -1);3.51(m, 2H, hexane CH₂ -6); 7.1-7.8 (m, 5H, aromatic); 8.16(s, 1H, imineCH). To a mixture of 2 g (0.01 mol) of 22 and 1.4 mL (0.01 mol) oftriethylamine in 20 mL of methylene chloride. Then 1.78 g (0.01 mol) ofbenzylchloroformate was added dropwise at -5° C. The resulting mixturewas stirred for 0.5 hr at 0° C. and for 2 hr at room temperature thendiluted to 50 mL with methylene chloride and washed with water (20 ml),2% sodium bicarbonate (20 ml), water and saturated sodium chloride anddried over anhydrous magnesium sulfate. After evaporation of solventunder reduced pressure the residue was dissolved in 5 mL of ethanol and10 mL of 2N hydrochloric acid was added. The resulting mixture wasstirred for 6 hr at room temperature then evaporated to dryness underreduced pressure. The residue was diluted to 50 mL with water and washedwith ethyl ether (2×15 ml). The water phase was evaporated under reducedpressure and the product 23 was purified by crystallization from a smallportion of water with a yield of 42%; mp 175°-178° C.

To prepare the dipeptide analog(N-(L-2-isobutyl-3-methoxycarbonyl)-propanoyl-L-tryptophan (25A)), forderivatization to 23: To a mixture of 1.754 g (9.32 mmol) of2-isobutyl-3-methoxycarbonylpropionic acid 3 in 4 mL of 50% anhydrousDMF in methylene chloride 1.66 g (10.2 mmol) of N,N'-carbonyldiimidazole(CDI) was added at room temperature. After 15 minutes of stirring atroom temperature, 3.08 g (9.31 mmol) of the hydrochloride salt ofL-tryptophan benzyl ester was added. The resulting mixture was stirredovernight at room temperature, then diluted to 60 mL with ethyl acetateand washed with 5% sodium bicarbonate (2×15 ml), water (2×15 ml),saturated sodium chloride solution and dried over magnesium sulfate.Evaporation of the solvent under reduced pressure gave 4.32 g (100%yield) of 24, the benzyl ester of 25 as a colorless foam, which was usedin the next step without further purification.

Hydrogen gas was bubbled through a mixture of 4.32 g (9.31 mmol) of 24and 0.5 g of 10% palladium on carbon in 15 mL of methanol for 2 hr whilemethanol was added to keep the volume of the reaction mixture constant.The catalyst was filtered off and washed with a fresh portion ofmethanol (15 ml) and the filtrate was evaporated to dryness underreduced pressure. Evaporation of the solvent under reduced pressure anddrying of the residue in vacuo gave 3.08 g (88% yield) of acid 25A,B asa mixture of two diastereoisomers, in the form of a colorless glassysolid. This was separated to give isomers 25A and 25B by flashchromatography (silica gel; ethyl acetate; R_(f) (25A)=0.24, R_(f)(25B)=0.1).

The compound 25A was converted toN-[L-2-isobutyl-3-methoxycarbonylpropanoyl]-L-tryptophan(6-phenylmethoxycarbonylamino-hexyl-1)amide (26A) as follows. A mixtureof 0.55 g (1.47 mmol) of 25A and 0.24 g (1.48 mmol) of CDI in 1 mL of 2%dimethylformamide in methylene chloride was stirred for 0.5 hr at roomtemperature and 0.42 g (1.47 mmol) of 23 was added. After stirringovernight at room temperature, the mixture was diluted to 50 mL withchloroform and washed with 2% potassium bisulfate (2×10 ml), water (10ml), 5% sodium bicarbonate (2×10 ml), water (2×10 ml) and saturatedsodium chloride and dried over anhydrous magnesium sulfate. Evaporationof the solvent under reduced pressure gave 0.8 g of the crude 26A whichwas purified by flash chromatography (silica gel; ethyl acetate/hexane25:5): Yield 56%; R_(f) (E)=0.57.

When the product 26A is substituted for 4A in Example 1, the identicalprocess afforded the title compound 27A, melting at 102°-108° C., in 46%yield; R_(f) (D)=0.63.

Example 11 Preparation ofN-[L-2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophancyclohexylamide (28A)

When cyclohexylamine is substituted for 23 in Example 10, the identicalprocess afforded the title compound 28A melting at 199°-203° C., in 49%yield; R_(f) (D)=0.51.

Example 12

Preparation ofN-[cis-2-(N'-hydroxycarbonylamido)-cyclohexylcarbonyl]-L-tryptophanmethylamide (29A,B)

A mixture of 2 g (0.013 mol) of cis-1,2-cyclohexane-dicarboxylicanhydride in 15 mL of methanol was refluxed for 5 hr, then evaporated todryness under reduced pressure to give 2.41 g (100% yield) ofcis-2-methoxycarbonyl-cyclohexanecarboxylic acid. When this wassubstituted for 3 in Example 1, the identical process afforded the titlecompound, melting at 140°-144° C., in 36% yield; R_(f) (D)=0.53, 0.47.

Example 13 Preparation ofN-trans-2-(N'-hydroxycarbonylamido)-cyclohexylcarbonyl]-L-tryptophanmethylamide (30A,B)

When (±)trans-1,2-cyclohexanedicarboxylic anhydride was substituted forcis-1,2-cyclohexanedicarboxylic anhydride in Example 12, the identicalprocess afforded the title compound 30A,B, melting at 167°-174° C., in37% yield; R_(f) (D)=0.57.

Example 14 Preparation ofN-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan (31A)

31A was prepared from 25A in Example 10 in a similar manner to thepreparation of 5A in Example 1 in 75% yield (128 mg) and isolated as afoam from ethyl acetate: R_(f) (F)=0.55, MS-FAB (m/z) (M⁺ +H). A smallsample of 31A recrystallized from ethyl acetate had a melting point of116°-120° C.

Example 15 Preparation ofN-(D,L-2-isobutyl-3-carboxypropanoyl)-L-tryptophan (6-aminohexyl-1)amide(32A)

A mixture of 0.5 g (8.24 mmol) of 26A in 0.4 mL of 2N potassiumhydroxide in methanol was stirred overnight at room temperature, thenevaporated to dryness under reduced pressure. The residue was diluted to15 mL with water and acidified to pH=2 with 1N hydrochloric acid. Thecrude free acid of 26A was taken up with ethyl acetate (3×15 ml) and theorganic phase was dried over anhydrous magnesium sulfate and evaporatedto dryness to give 0.45 g (92% yield) of 26A as a colorless foam.

Hydrogen gas was bubbled through a mixture of 0.395 g (6.6 mmol) of thefree acid of 26A in 15 mL of methanol for 2 hr, in the presence of 0.12g of 10% palladium on carbon at room temperature. The catalyst wasfiltered off, washed with ethanol (2×20 ml) and the filtrate wasevaporated to dryness under reduced pressure to give 0.3 g (92% yield)of the title compound 32A as a colorless foam; R_(f) (G) =0.08.

Example 16 Preparation ofN-[N-(2-isobutyl-3-carboxypropanoyl)-L-tryotophanyl] glycine 34A,B

The reaction of L-tryptophanyl-glycine methyl ester with acid 3,performed as described for 25A gave crudeN-[N-(D,L-2-isobutyl-3-methoxycarbonylpropanoyl)-L-tryptophanyl]-glycinemethyl ester 33 in 87% yield as a mixture of diastereomers 33A and 33B.Isomers 33A and 33B were separated by flash chromatography (silica gel;ethyl acetate). Isomer 33A mp =154°-155° C.; R_(f) (C)=0.46.

Esters 33A,B were transformed to free acids 34A,B by saponification withtwo equivalent of methanolic potassium hydroxide, as described for 25A.Isomer 34A yield 92%; mp=96°-102° C.; R_(f) (G)=0.31.

Isomer 34B yield 93%; mp=99°-105° C.; R_(f) (G)=0.25.

Example 17 Preparation ofN-(cis-2-carboxy-cyclohexylcarbonyl)-L-tryptophan methylamide 35

To a mixture of 0.281 g (1.82 mmol) of cis-1,2-cyclohexanedicarboxylicanhydride and 0.47 g of the hydrochloride salt of L-Trp-NHMe in 0.5 mLof dimethylformamide 0.51 mL of triethylamine was added at roomtemperature. After 2 hr of stirring the resulting mixture was diluted to10 mL with water and 25 mL of ethyl acetate was added. The resultingmixture was acidified to pH =2 with lo% potassium bisulfate and theorganic phase was washed with water (2×15 ml), saturated sodium chlorideand dried over anhydrous magnesium sulfate and evaporated to dryness.The title compound 35 was purified by crystallization from an ethylacetate-hexane mixture. Yield 48%; mp=105°-112° C.; R_(f) (G)=0.65,0.61.

Example 18 Preparation ofN-(trans-2-carboxy-cyclohexylcarbonyl)-L-tryptophan methylamide 36

When (±) trans-1,2-cyclohexanedicarboxylic anhydride is substituted forcis-1,2-cyclohexanedicarboxylic anhydride in Example 17, the identicalprocess afforded the title compound 36 in 56% yield: mp=167°-174° C.;R_(f) (G)=0.67, 0.61.

Example 19 Preparation ofN-[2-isobutyl-3-(N'-acetoxycarbonylamido)-propanoyl]-L-tryptophanmethylamide (37A)

To 97.5 mg (0.25 mmol) of 5A (Example 1) in 0.5 ml of dimethylformamidewas added 25.5 mg (0.25 mmol) of acetic anhydride and 37 mg (0.25 mmol)of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) at room temperature. Afterstanding overnight, the DMF was evaporated under high vacuum and theresidue taken up in a mixture of equal volumes of ethyl acetate and 2%potassium bisulfate. The ethyl acetate layer was washed with 2%potassium bisulfate, water, and brine, dried over magnesium sulfate, andevaporated to give a solid. The solid was dissolved in a 1:1 mixture ofhot ethyl acetate:hexane, which upon standing at room temperature gave71 mg (66% yield) of solid product 37A: mp=184°-186° C.; R_(f) (G)=0.68.

Example 20 Preparation ofN=[isobutyl-3-(N'-benzoxycarbonylamido)-propanoyl]-L-tryptophanmethylamide (38A)

To 30.5 mg (0.25 mmol) of benzoic acid in 1 ml of tetrahydrofuran wasadded 40.5 mg (0.25 mmol) of carbonyldiimidazole. After 10 minutes, 97mg (0.25 mmol) of compound 5A from Example 1 was added in 1 ml ofdimethylformamide. After 10 minutes, the reaction mixture was evaporatedto dryness under high vacuum, and dissolved in a mixture of equalvolumes of ethyl acetate and water. The ethyl acetate layer was washedwith 5% sodium bicarbonate, water, 2% sodium bisulfate, water, andbrine, and dried over magnesium sulfate. Evaporation of the ethylacetate layer to a small volume gave 50 mg (41%) of the title compound,38A: mp=187°-187.5° C.; Fr(G)=0.54.

Example 21

Applying the methods set forth above, the following invention compoundsare synthesized.

EtONHCONMe-CH₂ CH(iBu)-CO-L-Trp-NHEt

EtCONOH-CH₂ CH(iBu)-CO-L-Trp-NHEt

n-PrCONOEt-CH₂ CH(iBu)-CO-L-Trp-NHEt

EtNHCONOMe-CH₂ CH(iBu)-CO-L-Trp-NHEt

MeNHCONOH-CH₂ CH(iBu)-CO-L-Trp-NHEt

EtONHCONMe-CH₂ CH(iBu)-CO-L-Ala(2-naphthyl)- NHEt

EtCONOH-CH₂ CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt

n-PrCONOEt-CH₂ CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt

EtNHCONOMe-CH₂ CH(iBu)-CO-L-Ala(2-naphthyl)- NHEt

MeNHCONOH-CH₂ CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt

HONHCONHCH₂ CH(iBu)-CO-L-TrpNHMe

HONHCONHCH2CH₂ CH(iBu)-CO-L-TrpNHMe

HONHCONHCH(iBu)CO-L-TrpNHMe

H₂ NCON(OH)CH(iBu)CO-L-TrpNHMe

H₂ NCON(OH)CH₂ CH(iBu)CO-L-TrpNHMe

H₂ NCON(OH)CH2CH₂ CH(iBu)CO-L-TrpNHMe

CH₃ CON(OH)CH(iBu)CO-L-TrpNHMe

CH₃ CON(OH)CH₂ CH(iBu)CO-L-TrpNHMe

CH₃ CON(OH)CH₂ CH₂ CH(iBu)CO-L-TrpNHMe

Example 22 Assay of Inhibition Activity

Inhibitors were assayed against crude or purified human skin fibroblastcollagenase using the synthetic thiol ester substrate at pH 6.5 exactlyas described by Kortylewicz & Galardy, J Med Chem (1990) 33:263-273. Thecollagenase concentration was 1-2 nM. The compounds of Examples 1-18 aretested for their ability to inhibit crude collagenase and gelatinasefrom human skin fibroblasts, crude collagenase and gelatinase frompurulent human sputum in this assay. The results with respect to crudeenzyme preparation are shown in Table 1. The Ki of 5A for purified humanskin collagenase is 0.4 nM. Assays for inhibition of human stromelysinare conducted as described by Teahan, J., et al., Biochemistry (1989)20:8497-8501.

                                      TABLE 1                                     __________________________________________________________________________    No.   Compound                   K.sub.i (nM)                                 __________________________________________________________________________     1                                                                               5A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNHMe                                                                      10                                            1                                                                               5B NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNHMe                                                                      150                                           2                                                                               7A NHOHCOCH.sub.2 CH(i-Bu)CO-D-TrpNHMe                                                                      70,000                                        3                                                                               9A NHOHCOCH.sub.2 CH(i-Bu)CO-L-NMeTrpNHMe                                                                   500                                           4                                                                              11A NHOHCOCH.sub.2 CH(i-Bu)CO-L-Ala(2-naphthyl)NHMe                                                          15                                            5                                                                              13A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNH(CH.sub.2).sub.2 OH                                                     20                                            6                                                                              15A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNH(CH.sub.2).sub.4 CH.sub.3                                               30                                            7                                                                              17A, B                                                                            NHOHCOCH.sub.2 CH(i-Bu)CO-L-Trp-piperidine                                                               200                                           8                                                                              19A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNH(CH.sub.2).sub.11 CH.sub.3                                              300                                           9                                                                              21A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNH(S)CHMePh                                                               3                                            10                                                                              27A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNH(CH.sub.2).sub.6 NH-CBZ                                                 13                                           11                                                                              28A NHOHCOCH.sub.2 CH(i-Bu)CO-L-TrpNHcyclohexyl                                                              50                                           12                                                                              29A, B                                                                             ##STR5##                  >10,000                                      13                                                                              30A, B                                                                             ##STR6##                  >10,000                                      14                                                                              31A NHOHCO-CH.sub.2 CH(i-Bu)CO-L-TrpOH                                                                       200                                          15                                                                              32A HOOC-CH.sub.2 CH(i-Bu)CO-L-TrpNH(CH.sub.2)NH.sub.2                                                       >10,000                                      16                                                                              34A HOCO-CH.sub.2 CH(i-Bu)CO-L-Trp-Gly-OH                                                                    >10,000                                        34B HOCO-CH.sub.2 CH(i-Bu)CO-L-Trp-Gly-OH                                                                    >10,000                                      17                                                                              35                                                                                 ##STR7##                  >10,000                                      18                                                                              36                                                                                 ##STR8##                  >10,000                                      __________________________________________________________________________

Example 21 Prevention of Corneal Ulceration in the Alkali Burned RabbitCornea

The ability of the invention compounds to prevent ulceration has beenconfirmed by a corneal assay conducted by Gregory Schultz, University ofFlorida, Gainesville, Fla.

Twenty rabbit eyes were burned for 60 seconds with 1N NaOH to a diameterof 10 mm. The ten control eyes were treated with two drops of hypotonicbuffer every two hours from 8 AM to 6 PM, and then with asubconjunctival injection of 0.5 mL of buffer in the evening. Theexperimental eyes were treated identically but with 400 μg per mLinhibitor in buffer. Eyes were scored clinically with 0 being noulceration and 5 equalling perforation of the cornea. FIG. 1 showsaverage clinical score over 26 days. The compound 5A shows markedprotection of the cornea from perforation. FIG. 2 shows percent ofcorneas not perforated over 26 days; compound 5A shows 100% protection.

A histological examination of the treated and untreated corneas showedthe following:

Perpendicular sections though the corneas of rabbits were examined 28days following severe alkali injuries which were made by exposing thecorneas of anesthesized rabbits to 2 N sodium hydroxide for 60 secondsin a 12.5 mm diameter well. Following injury, rabbits were treatedtopically with 2 drops every other hour between 8 AM and 6 PM followedby a subconjunctival injection of 0.5 mL of collagenase inhibitor offormula 5A or vehicle (50 mM Hepes, Antibiotics).

The cornea of a rabbit treated with collagenase inhibitor shows lamellaethat have begun to repopulate with keratocytes which have most likelymigrated from the uninjured peripheral cornea and sclera. The stromaalso contains some inflammatory cells presumably macrophages which havemigrated into the injured cornea. There is little evidence fordisruption of the extracellular matrix of the stromal lamellae and thereis no evidence of significant extracellular matrix destruction. Theepithelium in this section has resurfaced the cornea, although theepithelium is tenuously attached as evidenced by the separation of thesections of the epithelium from the underlying stroma. There is noevidence of neovascularization of this part of the cornea. Theendothelial surface has not regenerated and Descemet's membrane hasseparated from the stroma in a cloudy cornea which lacks persistent andcomplete epithelial regeneration and also lacks significant stromalulceration.

Cornea treated only with vehicle shows a dominant appearance ofextensive degradation of the stromal matrix and the presence of massiveinflammatory cell infiltrates. These inflammatory cells most probablyare macrophages and neutrophils. The stromal lamellae have digested inthis section approximately two-thirds of the complete depth of thestroma, and in adjacent sections, erosion has occurred to Descemet'smembrane. The stroma appears to have a frayed appearance at the edgewhere the inflammatory cell infiltration is most extensive. Fracturelines running through the stroma suggest a general weakening of theextracellular matrix. In this section of the cornea there is no evidenceof neovascularization, epithelial cells, or endothelial cells. Fragmentsof endothelial cells are present on Descemet's membrane together withinflammatory cells in the anterior chamber fluid. Few if any keratocytescan be identified in the stroma. This microscopic section is generallyconsistent with the results of slit lamp microscopy which indicated thatthe cornea had extensive ulceration and peripheral neovascularization.

Overall, the histopathology of these two sections suggest that a majoreffect of the collagenase inhibitor in preventing ulceration is due to areduction in the inflammatory cell filtration into the alkali injuredcornea. Furthermore, this section suggests that repopulation of thestroma in the collagenase inhibitor treated corneas has begun andincomplete epithelial regeneration of a transient nature has begun onthe epithelial surface with no regeneration of the endothelium.

We claim:
 1. A compound of the formula ##STR9## and wherein R⁴ is(3-indolyl)methylene, and the pharmaceutically acceptable amidesthereof.
 2. The compound of claim 1 which is selected from the groupconsisting ofHONHCONHCH₂ CH(i-Bu)CO-L-Trp-NHMe; HONHCONHCH₂CH(i-Bu)CO-L-Trp-NH(CH₂)₂ OH; HONHCONHCH₂ CH(i-Bu)CO-L-Trp-NH(CH₂)₄ CH₃; HONHCONHCH₂ CH(i-Bu)CO-L-Trp-piperidine; HONHCONHCH₂CH(i-Bu)CO-L-Trp-(CH₂)₁₁ CH₃ ; HONHCONHCH₂ CH(i-Bu)CO-L-Trp-NHCHMePh;HONHCONHCH₂ CH(i-Bu)CO-L-Trp-NH(CH₂)₆ NH-CBZ; HONHCONHCH₂CH(i-Bu)CO-L-Trp-NHcyclohexyl; and HONHCONHCH₂ CH(i-Bu)CO-L-Trp-OH. 3.The compound of claim 2 which is selected from the group consistingofHONHCONHCH₂ CH(i-Bu)CO-L-Trp-NHMe; HONHCONHCH₂CH(i-Bu)CO-L-Trp-NH(CH₂)₂ OH; HONHCONHCH₂ CH(i-Bu)CO-L-Trp-NH(CH₂)₄ CH₃; HONHCONHCH₂ CH(i-Bu)CO-L-Trp-NHCHMePh; and HONHCONHCH₂CH(i-Bu)CO-L-Trp-NH(CH₂)₆ NH-CBZ.
 4. The compound of claim 2 which isHONHCONHCH₂ CH(i-Bu)CO-L-Trp-NHMe.